The present invention relates to compositions and methods for the prevention and treatment of blood-borne and toxin-mediated diseases, and in particular anti-C5a antibodies for the prevention and treatment of sepsis in humans as well as other animals.
Sepsis is a major cause of morbidity and mortality in humans and other animals. It is estimated that 400,000-500,000 episodes of sepsis resulted in 100,000-175,000 human deaths in the U.S. alone in 1991. Sepsis has become the leading cause of death in intensive care units among patients with non-traumatic illnesses. [G. W. Machiedo et al., Surg. Gyn. and Obstet. 152:757-759 (1981).] It is also the leading cause of death in young livestock, affecting 7.5-29% of neonatal calves [D. D. Morris et al., Am. J. Vet. Res. 47:2554-2565 (1986)], and is a common medical problem in neonatal foals. [A. M. Hoffinan et al., J. Vet. Int. Med. 6:89-95 (1992).] Despite the major advances of the past several decades in the treatment of serious infections, the incidence and mortality due to sepsis continues to rise. [S. M. Wxc3x3lff, New Eng. J. Med. 324:486-488 (1991).]
Sepsis is a systemic reaction characterized by arterial hypotension, metabolic acidosis, decreased systemic vascular resistance, tachypnea and organ dysfunction. Sepsis can result from septicemia (i.e., organisms, their metabolic end-products or toxins in the blood stream), including bacteremia (i.e., bacteria in the blood), as well as toxemia (i.e., toxins in the blood), including endotoxemia (i.e., endotoxin in the blood). The term xe2x80x9cbacteremiaxe2x80x9d includes occult bacteremia observed in young febrile children with no apparent foci of infection. The term xe2x80x9csepsisxe2x80x9d also encompasses fungemia (i.e., fungi in the blood), viremia (i.e., viruses or virus particles in the blood), and parasitemia (i.e., helminthic or protozoan parasites in the blood). Thus, septicemia and septic shock (acute circulatory failure resulting from septicemia often associated with multiple organ failure and a high mortality rate) may be caused by a number of organisms.
The systemic invasion of microorganisms presents two distinct problems. First, the growth of the microorganisms can directly damage tissues, organs, and vascular function. Second, toxic components of the microorganisms can lead to rapid systemic inflammatory responses that can quickly damage vital organs and lead to circulatory collapse (i.e., septic shock) and oftentimes, death.
There are three major types of sepsis characterized by the type of infecting organism. Gram-negative sepsis is the most common and has a case fatality rate of about 35%. The majority of these infections are caused by Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Gram-positive pathogens such as the Staphylococci and Streptococci are the second major cause of sepsis. The third major group includes fungi, with fungal infections causing a relatively small percentage of sepsis cases, but with a high mortality rate.
Many of these infections are acquired in a hospital setting and can result from certain types of surgery (e.g., abdominal procedures), immune suppression due to cancer or transplantation therapy, immune deficiency diseases, and exposure through intravenous catheters. Sepsis is also commonly caused by trauma, difficult newborn deliveries, and intestinal torsion (especially in dogs and horses).
Many patients with septicemia or suspected septicemia exhibit a rapid decline over a 24-48 hour period. Thus, rapid methods of diagnosis and treatment delivery are essential for effective patient care. Unfortunately, a confirmed diagnosis as to the type of infection traditionally requires microbiological analysis involving inoculation of blood cultures, incubation for 18-24 hours, plating the causative organism on solid media, another incubation period, and final identification 1-2 days later. Therefore, therapy must be initiated without any knowledge of the type and species of the pathogen, and with no means of knowing the extent of the infection.
It is widely believed that anti-endotoxin antibody treatment administered after sepsis is established may yield little benefit because these antibodies cannot reverse the inflammatory cascade initiated by endotoxin. In addition, the high cost of each antibody could limit physicians"" use of a product where no clear benefit has been demonstrated. [K. A. Schulman et al., JAMA 266:3466-3471 (1991).] Furthermore, these endotoxin antibodies only target gram-negative sepsis, and no equivalent antibodies exist for the array of gram-positive organisms and fungi.
Clearly, there is a great need for agents capable of preventing and treating sepsis. It would be desirable if such agents could be administered in a cost-effective fashion. Furthermore, approaches are needed to combat all forms of sepsis.
The present invention relates to compositions and methods for the prevention and treatment of blood-borne and toxin mediated diseases, and in particular anti-C5a antibodies for the prevention and treatment of sepsis in humans as well as other animals.
The present invention provides a composition comprising antibody specific for complement component C5a peptide. In another embodiment, the composition comprises antibody which is specific for complement component C5a peptide, wherein the C5a peptide has a C-terminal region and an N-terminal region, and the antibody is not reactive with the C-terminal region. In further embodiments, the antibody is specific for the N-terminal region of complement component C5a peptide. In an additional embodiment, the antibody is also not reactive with complement component C5 protein.
It is not intended that the present invention be limited to antibodies specific for C5a peptides from certain animals. In certain embodiments, the antibody is specific for rat C5a peptide. In other embodiments, the antibody is specific for bovine C5a peptide. In still other embodiments, the antibody is specific for porcine C5a peptide. In a preferred embodiment, the antibody is specific for human C5a peptide.
It is also not intended that the present invention be limited to antibodies generated in a particular animal. A variety of animals are useful for generating the antibodies of the present invention. In one embodiment, the antibody is generated in an animal selected from a mouse, a rat, a horse, a goat, a chicken, and a rabbit. In some embodiments, the antibodies are collected from the blood of the animal. In other embodiments, the animal generating the antibodies is a bird, and the antibodies are collected from egg yolk.
It is not intended that the present invention be limited to the nature of the antibodies, as a variety of antibody types are contemplated. In one embodiment, the antibodies are monoclonal. In another embodiment, the antibodies are humanized. In other embodiments, the antibodies are chimaeric. In a preferred embodiment, the antibodies are polyclonal.
The present invention also provides a method of producing polyclonal antibody. In one embodiment, the method comprises, providing; an animal and an immunogenic composition, wherein the composition comprises C-terminal truncated C5a peptides; and immunizing the animal with the immunogenic composition in order to generate antibodies. In some embodiments, the immunogenic composition comprises adjuvant. In a further embodiment, antibodies are collected from the animal.
It is not intended that the present invention be limited to antibodies specific for C5 a peptides from any particular animal. In certain embodiments, the antibody is specific for rat C5a peptide. In other embodiments, the antibody is specific for bovine C5a peptide. In still other embodiments, the antibody is specific for porcine C5a peptide. In a preferred embodiment, the antibody is specific for human C5a peptide.
It is not intended that the present invention be limited to particular C-terminal truncated peptides. A variety of C-terminal truncated peptides are contemplated. In one embodiment, the C-terminal truncated peptide corresponds to the entire N-terminal region of C5a peptide. In another embodiment, the C-terminal truncated peptide corresponds to the entire N-terminal region of C5a peptide and a portion of the C-terminal region. In another embodiment, the C-terminal truncated peptide is a fragment or portion of the N-terminal region of C5a peptide. In another embodiment, the C-terminal truncated C5a peptide is between approximately 5 and 50 amino acids in length. In some embodiments, the C-terminal truncated peptide is approximately fifty amino acids in length. In other embodiments, the C-terminal truncated peptide is approximately five amino acids in length. In preferred embodiments, the C-terminal truncated peptides are 20 amino acids in length. In certain embodiments, the C-terminal truncated peptides are selected from SEQ ID NOS:2, 4, 5, 14, 15, and 16.
The present invention also provides a method of treating a subject with the antibodies of the present invention. In one embodiment, the method comprises; providing; a subject, and a therapeutic composition comprising an antibody specific for complement component C5a peptide, wherein the C5a peptide has a C-terminal region and an N-terminal region, and wherein the antibody is not reactive with the C-terminal region; and administering the therapeutic composition to the subject. In another embodiment, the antibody is specific for the N-terminal region of complement component C5a peptide.
In one embodiment, the present invention provides a method comprising; providing; a subject, and a therapeutic composition comprising an antibody specific for complement component C5a peptide, wherein the C5a peptide has a C-terminal region and an N-terminal region, and wherein the antibody is not reactive with the C-terminal region; and administering the therapeutic composition to the subject. In another embodiment, administering the therapeutic composition reduces the binding of complement component C5a peptide to one or more neutrophils of the subject. In a certain embodiment, administering the therapeutic composition reduces bacteremia in the subject. In yet another embodiment, administering the therapeutic composition increases the H2O2 production of neutrophils of the subject. In a preferred embodiment, administering the therapeutic composition reduces the symptoms of sepsis.
It is not intended that the therapeutic method of the present invention be limited to particular subjects. A variety of subjects are contemplated. In one embodiment the subject is selected from a pig, a rat, a cow, a horse, and a human. In one embodiment, the therapeutic composition is administered to a subject suffering from symptoms of sepsis. In another embodiment, the therapeutic composition is administered prophylactically to a subject at risk for sepsis, including new born humans and animals.
It is not intended that the therapeutic method of the present invention be limited to certain modes of administration. A variety of modes of administering the therapeutic composition are contemplated. In one embodiment, the therapeutic composition is administered by a mode selected from intravenously, intra-muscularly, subcutaneously, intradermally, intraperitoneally, intrapleurally, intrathecally, and topically.
It is not intended that the present invention be limited to a particular therapeutic composition. A variety of compositions are contemplated. In one embodiment the therapeutic composition comprises a soluble mixture of anti-C5a antibodies. In another embodiment, the anti-C5a antibodies are provided together with physiologically tolerable liquid, gels, solid carriers, diluents, adjuvants or excipients, and combinations thereof. In other embodiments, the therapeutic composition comprises anti-C5a antibodies and other therapeutic agents (e.g. other immunoglobulins or antibiotics).
The present invention also provides a method for screening C-terminal truncated C5a peptides to identify immunogens for the production of anti-C5a antibodies. In one embodiment, the method comprises, providing a C-terminal truncated C5a peptide, modifying the amino acid sequence of said C-terminal truncated C5a peptide, and screening said C-terminal truncated C5a peptide to identify immunogens for the production of anti-C5a antibodies. In one embodiment, the C-terminal truncated C5a peptide which is provided is selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:14, SEQ ID NO: 15, and SEQ ID NO:16. In other embodiments, the screening step involves a chemotaxis assay (See e.g. Examples 7, 8 and 11). In a different embodiment, the screening step involves a competitive binding assay (See e.g. Examples 10 and 11). In an additional embodiment, the screening step involves administering the C-terminal truncated peptides to septic animals (See e.g. Example 11).